Document Type

Article

Publication Date

10-25-1990

Publisher

Elsevier

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Original Item ID

DOI: 10.1016/S0021-9258(17)44801-0

Abstract

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains (“A” and “B,” respectively) in the central portion of the molecule, and exposure of a noncatalytic “secondary” thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the Bβ 1-42 sequence (“des-(Bβ 1-42)-fibrinogen”), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by Bβ 1-42 in the polymerization of α-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (μ, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(Bβ 1-42)-α-fibrin, thus indicating that the Bβ 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(Bβ 1-42)-α-fibrin polymerized even more slowly than thrombin-activated des-(Bβ 1-42)-α-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent α-fibrin monomers. Thrombin-activated des-(Bβ 1-42)-α-fibrin polymerized more rapidly at low ionic strength (μ, 0.04) than did native α,β-fibrin, a reversal of their behavior at physiological ionic strength (μ, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(Bβ 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native α,β-fibrin at low ionic strength, coupled with weak thrombin binding to des-(Bβ 1-42)-α-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(Bβ 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B15-42 sequence in fibrin plays a major role in forming or providing this site.

Comments

Published version. Journal of Biological Chemistry, Vol. 265, No. 30 (October 25, 1990): 18650-18655. DOI. © 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

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